Preparation of (S)-N-Substituted 4-Hydroxy-pyrrolidin-2-ones by Regio- and Stereoselective Hydroxylation with Sphingomonas sp. HXN-200

Article abstract of DOI:10.1021/ol006735q

formula presented Enantiopure (S)-N-substituted 4-hydroxy-pyrrolidin-2-ones have been prepared for the first time by regio- and stereoselective hydroxylation of the corresponding pyrrolidin-2-ones by use of a biocatalyst. Hydroxylation of 6 and 8 with Sph

Full text of DOI:10.1021/ol006735q

ORGANIC
LETTERS
2000
Vol. 2, No. 24
3949-3952
Preparation of (S)-N-Substituted
4-Hydroxy-pyrrolidin-2-ones by Regio-
and Stereoselective Hydroxylation with
Sphingomonas sp. HXN-200
Dongliang Chang, Bernard Witholt, and Zhi Li*
Institute of Biotechnology, ETH-Zurich, Honggerberg, CH-8093 Zurich, Switzerland.
zhi@biotech.biol.ethz.ch
Received October 16, 2000
ABSTRACT
Enantiopure (S)-N-substituted 4-hydroxy-pyrrolidin-2-ones have been prepared for the first time by regio- and stereoselective hydroxylation of
the corresponding pyrrolidin-2-ones by use of a biocatalyst. Hydroxylation of 6 and 8 with Sphingomonas sp. HXN-200 afforded 68% of (S)-7
in >99.9% ee and 46% of (S)-9 in 92% ee, respectively. Simple crystallization increased the ee of (S)-9 to 99.9% in 82% yield.
Optically active 4-hydroxy-pyrrolidin-2-one and its N-
substituted derivatives are useful intermediates for the
preparation of several pharmaceuticals. The (S)-enantiomers,
for example, can be used in the synthesis of an oral
carbapenem antibiotic CS-8341 1 and nootropic drug (S)-
Oxiracetame2 2; the (R)-enantiomers can be used in the
preparation of an antidepressant agent (R)-Rolipram3 3,
been developed, but each has one or more drawbacks: (1)
Syntheses via direct cyclization4c,6 or cyclization with am-
monia7 or with alkyl- or aralkylamine8 need optically active
precursors that cannot be prepared easily. (2) Preparation
involving reduction of (S)-N-benzyl-4-hydroxy-pyrrolidin-
2,5-dione9 is multistep and requires special reagents. (3)
Synthesis from (2S,4R)-4-hydroxyproline10 requires expen-
anticonvulsant
(R)-γ-amino-â-hydroxybutyric
acid
(GABOB)4,5 4, and antihyperlipoproteinemic L-Carnitine
(6) (a) Kobayashi, S.; Kobayashi, K.; Hirai, K. Synlett 1999, S1, 909.
(b) Srairi, D.; Maurey, G. Bull. Soc. Chim. Fr. 1987, 2, 297. (c) Inagaki,
S.; Shitara, H.; Hirose, T.; Nohira, H. Enantiomer 1998, 3, 187. (d)
Mitsuhashi, S.; Sumi, K.; Moroi, T.; Sotoguchi, T.; Miura, T. EP 0947505
A2, 6 Oct 1999. (e) Nishikawa, M. EP 0844242 A1, 27 May 1998. (f)
Furukawa, Y.; Takenaka, K. JP 9031058 A2, 4 Feb 1997. (g) Kobayashi,
K.; Fukuhara, H.; Takebayashi, T.; Kawamoto, I. JP 8119935, 14 May 1996.
(h) Furukawa, Y.; Shiomi, Y.; Nagao, K. WO 9636603, 21 Nov 1996. (i)
Mitsuhashi, S.; Kumobayashi, H. EP 0787718 A1, 6 Aug 1997. (j)
Kawamoto, I.; Sugano, O.; Ishikawa, K.; Takebayashi, T. JP 8319271 A2,
3 Dec 1996.
(7) (a) Pellegata, R.; Dosi, I.; Villa, M.; Lesma, G.; Palmisano, G.
Tetrahedron 1985, 41, 5607. (b) Seki, M.; Kondo, K. Synthesis 1999, 5,
745. (c) Santaniello, E.; Casati, R.; Milani, F. J. Chem. Res., Synop. 1984,
132. (d) Osai, Y.; Ayabe, M.; Aoki, K.; Shimizai, T.; Kobayashi, I. JP
57183756 A2, 12 Nov 1982.
(vitamin BT)5 5.
Several methods for synthesis of optically active 4-hy-
droxy-pyrrolidin-2-one and its N-substituted derivatives have
(1) (a) Miyadera, T.; Sugimura, Y.; Hashimoto, T.; Tanaka, T.; Iino,
K.; Shibata, T.; Sugawara, S. J. Antibiot. 1983, 36, 1034. (b) Miyauchi,
M.; Endo, R.; Hisaoka, M.; Yasuda, H.; Kawamoto, I. J. Antibiot. 1997,
50, 429.
(2) (a) Banfi, S.; Fonio, W.; Allievi, E.; Pinza, M.; Dorigotti, L. Farmaco
Ed. Sci. 1984, 39, 16. (b) Almeida, J. F.; Anaya, J.; Martin, N.; Grande,
M.; Moran, J. R.; Carballero, M. C. Tetrahedron: Asymmetry 1992, 3, 1431.
(3) Mulzer, J.; Zuhse, R.; Schmiechen, R. Angew. Chem., Int. Ed. Engl.
1992, 31, 870.
(4) (a) Jung, M. E.; Shaw, T. J. J. Am. Chem. Soc. 1980, 102, 6304. (b)
Krogsgaard-Larsen, P.; Nielsen, L.; Falch, E.; Curtis, D. R. J. Med. Chem.
1985, 28, 1612. (c) Orena, M.; Porzi, G.; Sandri, S. J. Chem. Res., Synop.
1990, 376.
(5) Aube, J.; Wang, Y.; Ghosh, S.; Langhans, K. L. Synth. Commun.
1991, 21, 693.
(8) Kawamoto, I.; Ishikawa, K.; Endo, R.; Takebayashi, T. JP 7324071
A2, 12 Dec 1995.
(9) Huang, P. Q.; Zheng, X.; Wang, S. L.; Ye, J. L.; Jin, L. R.; Chen, Z.
Tetrahedron: Asymmetry 1999, 10, 3309.
(10) Haeusler, J. Monatsh. Chem. 1987, 118, 865.
10.1021/ol006735q CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/10/2000

sive starting material. (4) Synthesis via photochemical
rearrangement of special oxaziridines5 occurs with low yield.
(5) Resolution of racemic 4-hydroxy-pyrrolidin-2-ones with
stereoselective esterase11 is a low-yield process and requires
the preparation of the racemates.
In our previous study on biohydroxylation of pyrrolidines,14a
we found that Sphingomonas sp. HXN-20015 is an excellent
biocatalyst for regio- and stereoselective hydroxylation of
N-substituted pyrrolidines, giving the corresponding optically
active 3-hydroxypyrrolidines. Here, we report a simple and
practical synthesis of (S)-N-substituted 4-hydroxy-pyrrolidin-
2-ones by hydroxylation of the corresponding pyrrolidin-2-
ones with Sphingomonas sp. HXN-200 as biocatalyst.
Hydroxylation of 6 and 8 was performed with resting cells
of Sphingomonas sp. HXN-200 on a 10-mL scale in the
exploratory stage.16 The reaction was followed by analytical
HPLC.17 Hydroxylation of 6 and 8 afforded the desired
4-hydroxy products 7 and 9, respectively. Comparison of
the retention time and the UV absorption area at 210 nm
with the standards of 6-9 suggested the conversion to the
products.
As shown in Table 1, hydroxylation of a 2 mM solution
of N-benzyl-pyrrolidin-2-one 6 with resting cells (4.0 g/L)
Regio- and stereoselective hydroxylation of pyrrolidin-2-
ones is the simplest route for preparing optically active
4-hydroxy-pyrrolidin-2-one and its N-substituted derivatives.
However, regio- and stereoselective hydroxylation on non-
activated carbon atom remains a challenge in synthetic
chemistry.12 On the other hand, biohydroxylation can be a
useful tool for this type of transformation.13,14 However,
selective biohydroxylation of pyrrolidin-2-ones has proven
to be very difficult. Hydroxylation of N-benzoyl- and
N-phenylacetyl-pyrrolidin-2-one with BeauVeria sulfurescens
(ATCC 7159), a well-known fungus for hydroxylation, gave
only 21% of N-benzoyl-4-hydroxy-pyrrolidin-2-one and 5%
of N-phenylacetyl-4-hydroxy-pyrrolidin-2-one, respectively,
in very low ee.6b Moreover, several byproducts were formed
in each case.
Table 1. Hydroxylation of 6 to 7 with Resting Cells (4.0 g/L)
of Sphingomonas sp. HXN-200
7 (%)
6
glucose
(%)
activitya
(mM)
(U/g CDW) 0.5 h
1 h
2 h
3 h 5 h
2.0
2.0
3.0
4.0
5.0
5.0
0
2
2
2
0
2
2.6
4.4
4.6
4.1
3.0
4.3
15
26
18
12
7.0
10
19
41
29
19
8.0
14
22
62
49
36
9.0
24
22
69
58
47
10
36
23
70
65
57
10
47
(11) Maeda, K.; Inukai, M. JP 10165195 A2, 23 June 1998.
(12) For reviews see: (a) Reiser, O. Angew. Chem., Int. Ed. Engl. 1994,
33, 69. (b) Barton, D. H. R.; Doller, D. Acc. Chem. Res. 1992, 25, 504. (c)
Ostovic, D.; Bruice, T. C. Acc. Chem. Res. 1992, 25, 314. (d) Davis, J. A.;
Watson, P. L.; Liebmann, J. F.; Greenberg, A. SelectiVe Hydrocarbon
ActiVation; VCH: New York, 1990. (e) Hill, C. L. ActiVation and
Functionalization of Alkanes; Wiley and Sons: New York, 1989. (f) Shilov,
A. V. ActiVation of Saturated Hydrocarbons by Transition Metal Complexes;
Reisel: Boston, 1984.
(13) For reviews see: (a) Johnson, R. A. In Oxidation in Organic
Chemistry; Trahanovsky, W. S., Ed.; Academic Press: New York, 1978;
Part C, p 131. (b) Kieslich, K. Microbial Transformation of non-Steroid
Cyclic Compounds, Thieme: Stuttgart, 1976; p 365. (c) Holland, H. L.
Steroids 1999, 64, 178. (d) Holland, H. L. Organic Synthesis with OxidatiVe
Enzymes; VCH: New York, 1992; Chapter 3, p 55. (e) Holland, H. L. Acc.
Chem. Res. 1984, 17, 389.
(14) For recent publications see: (a) Li, Z.; Feiten, H.-J.; van Beilen, J.
B.; Duetz, W.; Witholt, B. Tetrahedron: Asymmetry 1999, 10, 1323. (b)
Hemenway, M. S.; Olivo, H. F. J. Org. Chem. 1999, 64, 6312. (c) Holland,
H. L.; Morris, T. A.; Nava, P. J.; Zabic, M. Tetrahedron 1999, 55, 7441.
(d) Braunegg, G.; de Raadt, A.; Feichtenhofer, S.; Griengl, H.; Kopper, I.;
Lehmann, A.; Weber, H. J. Angew. Chem., Int. Ed. 1999, 38, 2763. (e)
Palmer, C. F.; Webb, B.; Broad, S.; Casson, S.; McCague, R.; Willetts, A.
J.; Roberts, S. M. Bioorg. Med. Chem. Lett. 1997, 7, 1299. (f) Pietz, S.;
Fro¨hlich, R.; Haufe, G. Tetrahedron 1997, 53, 17055. (g) Flitsch, S. L.;
Aitken, S. J.; Chow, C. S.-Y.; Grogan, G.; Staines, A. Bioorg. Chem. 1999,
27, 81. (h) Lutz-Wahl, S.; Fischer, P.; Schmidt-Dannert, C.; Wohlleben,
W.; Hauer, B.; Schmid, R. D. Appl. EnViron. Microbiol. 1998, 64, 3878.
(15) (a) Sphingomonas sp. HXN-200 was isolated from a waste air filter
by Plaggemeier, Th.; Schmid, A.; Engesser, K. at University of Stuttgart.
(b) For growth conditions, see ref 14a. (c) This strain is available for
scientific researches from the culture collection at Institute of Biotechnology,
ETH Zurich, Switzerland.
a Activity was determined over the first 30 min.
of Sphingomonas sp. HXN-200 that had been prepared by
using octane vapor as sole carbon source15b gave 70% of
the desired N-benzyl-4-hydroxy-pyrrolidin-2-one 7 as main
product18 in the presence of glucose (2%, w/v) for 5 h. The
addition of glucose increased the conversion significantly.
This is because the biohydroxylation is cofactor-dependent
and the addition of glucose contributed to the intracellular
regeneration of cofactors. This effect was also observed in
(16) General Procedure. Substrate 6 or 8 (2-16 mM) was added to 10
mL of cell suspension (4.0 g/L) of Sphingomonas sp. HXN-200 in 50 mM
potassium phosphate buffer (pH 8.0) containing glucose (0-2%, w/v) in a
100 mL shaking flask. The mixture was shaken at 200 rpm and 30 °C for
5 h. Samples (100 µL) were taken out at different times and mixed with
methanol (100 µL), and the cells were removed by centrifugation. The
supernatant was analyzed by HPLC.
(17) HPLC analysis: Hypersil BDS-C18 column (125 mm × 4 mm);
UV detection at 210 nm; acetonitrile/10mM potassium phosphate buffer
(pH 7.0) 20/80 as eluent; flow at 1 mL/min; retention time 2.7 min for 7,
8.1 min for 6, 2.7 min for 9, and 6.7 min for 8.
(18) N-Benzyl-3-hydroxy-pyrrolidin-2-one was formed as byproduct.
Ratio of 7/byproduct is about 5/1.
3950
Org. Lett., Vol. 2, No. 24, 2000

hydroxylation of a 5 mM solution of 6; the conversion to 7
at 5 h was increased from 10% to 47% by addition of 2% of
glucose. Hydroxylation of 3 and 4 mM solutions of 6 for 5
h gave 65% and 57% of 7, respectively, with activity of 4.6
and 4.1 U/g CDW (U ) µmol/min, CDW ) cell dry weight)
in the first 30 min.
Higher activity was observed for hydroxylation of N-tert-
butoxycarbonyl-pyrrolidin-2-one 8 with resting cells (4.0 g/L)
of Sphingomonas sp. HXN-200. As shown in Table 2,
isolated. For a practical bioconversion, the product concen-
tration has to be increased. This can be easily achieved by
using higher starting concentration of 6 and higher cell
density. Hydroxylation of a 6 mM solution of 6 with 8.0
and 10 g/L of resting cells of Sphingomonas sp. HXN-200
afforded 42% (0.48 g/L) and 68% (0.78 g/L) of 7, respec-
tively.
Biohydroxylation of 8 (14 mM) with 4.0 g/L of resting
cells of Sphingomonas sp. HXN-200 afforded 920b in 39%
yield (1.10 g/L). Similarly, increase of cell density to 8.0
g/L improved the yield to 1.29 g/L (46%). Further improve-
ment was achieved by use of more substrate and more cells:
hydroxylation of a 20 mM solution of 8 with 10 g/L of
resting cells of Sphingomonas sp. HXN-200 gave the pure
product 9 in 42% yield (1.69 g/L).
Table 2. Hydroxylation of 8 to 9 with Resting Cells (4.0 g/L)
of Sphingomonas sp. HXN-200
On the basis of our experience, the yield of 7 and 9 can
be further improved by performing the hydroxylation in a
bioreactor.
For determination of the ee of the biohydroxylation
products 7 and 9, standard (R)- and (S)-7 and 9 were
synthesized from the corresponding known compounds (R)-
and (S)-1021. As shown in Scheme 1, benzylation of (R)-
9 (%)
8a
activityb
(mM)
(U/g CDW)
0.5 h
1 h
2 h
3h
5h
2.0
5.0
8.0
10
14
16
4.4
6.8
8.9
8.5
11
26
16
13
10
9.0
7.0
47
34
28
22
22
19
67
54
48
39
40
34
73
66
61
52
51
45
80
76
71
65
63
57
Scheme 1
9.5
a Bioconversion was performed in the presence of glucose (2%).
b Activity was determined over the first 30 min.
hydroxylation of a 14 mM solution of 8 gave an activity of
11 U/g CDW and 63% conversion to 9 at 5 h. Interestingly,
both conversion and activity are not very much dependent
on the starting concentration of substrate, which is advanta-
geous for practical bioconversions; 57-80% of 9 were
formed in hydroxylations of 2-16 mM solutions of 8 for 5
h. No byproduct was formed in biohydroxylation of 8,
demonstrating the excellent regioselectivity of the biocatalyst.
Preparation of 7 and 9 were performed on a 50-mL scale.19
As shown in Table 3, biohydroxylation of 6 (3 mM) with
resting cells (4.0 g/L) of Sphingomonas sp. HXN-200 for 5
h formed 66% of 7; 55% (0.32 g/L) of pure product20a was
and (S)-10 afforded the corresponding (R)- and (S)-11 in 19%
and 23% yield, respectively. Deprotection of 11 gave (R)-
and (S)-7 in 41% and 34% yield, respectively. Similarly,
treatment of (R)- and (S)-10 with Boc2O, DMAP, and Et3N
(19) Preparation of 7. To 50 mL of cell suspension (4.0 g/L) of
Sphingomonas sp. HXN-200 in 50 mM potassium phosphate buffer (pH
8.0) containing glucose (2%, w/v) in a 500 mL shaking flask was added 6
(26.3 mg, 0.15 mmol). The mixture was stirred at 200 rpm and 30 °C. The
reaction was followed by analytical HPLC and was stopped at 5 h with
66% conversion. The cells were removed by centrifugation, and the product
was extracted into ethyl acetate. The organic phase was dried over Na2-
SO4, filtered, and evaporated. Purification by column chromatography on
silica gel (Rf of 7 ) 0.13 and Rf of 6 ) 0.50; ethyl acetate/methanol 9:1)
afforded 15.8 mg (55%) of 6 as white powder.
(M+1, 100), 174.1(9). IR (cm-1): 3401, 3007, 2928, 1682, 1483, 1435,
1262, 1082. (b) Data for 9. 1H NMR (300 MHz, CDCl3): δ 4.47 (s, 1 H,
H-C(4)), 3.88 (dd, 1 H, J ) 11.9 and 5.1 Hz, HA-C(5)), 3.77 (d, 1 H, J )
11.8 Hz, HB-C(5)), 3.15 (s, 1 H, OH), 2.77 (dd, 1 H, J ) 17.7 and 6.1 Hz,
HA-C(3)), 2.43 (d, 1 H, J ) 17.7 Hz, HB-C(3)), 1.52 ppm (s, 9 H, 3 CH3).
13C NMR (75 MHz, CDCl3): δ 172.8 (s, COO); 150.05 (s, CO); 83.19 (s,
C(CH3)3); 63.03 (d, C-4); 55.31 (t, C-5); 42.71 (t, C-3); 28.03 ppm (q,
CH3). MS: m/z 202 (M+1, 2), 146.0 (100), 128.0 (13), 113.0 (12), 102.1
(81). IR (cm-1): 3399, 2983, 1782, 1747, 1715, 1370, 1308, 1152, 1078,
(20) (a) Data for 7. Mp 107.3-108.0 °C. [R]25D -34.1° (c 1.00, CHCl3).9
1H NMR (300 MHz, CDCl3): δ 7.35-7.19 (m, 5 H, aromatic H), 4.52-
4.38 (m, 3 H, NCH2Ph, H-C(4)), 3.48 (dd, 1 H, J ) 10.9 and 5.6 Hz, HA-
C(5)), 3.26 (s, br., 1 H, OH), 3.18 (dd, 1 H, J ) 10.8 and 2.0 Hz, HB-
C(5)), 2.70 (dd, 1 H, J ) 17.4 and 6.6 Hz, HA-C(3)), 2.43 ppm (dd, 1 H,
J ) 17.3 and 2.5 Hz, HB-C(3)). 13C NMR (75 MHz, CDCl3): δ 172.94 (s,
CO); 135.97 (s), 128.70 (d), 127.98 (d), 127.60 (d, aromatic C); 64.27 (d,
C-4); 55.71 (t, CH2Ph); 46.32 (t, C-5); 41.14 ppm (t, C-3). MS: m/z 192.1-
1022, 848. (c) Data for 9 (after crystallization): mp 133.5-134.6 °C. [R]25
D
+2.1° (c 1.86, CHCl3).
(21) Kanno, O.; Miyauchi, M.; Shibayama, T.; Ohya, S.; Kawamoto, I.
J. Antibiot. 1999, 52, 900.
Org. Lett., Vol. 2, No. 24, 2000
3951

afforded 89% and 92% of (R)- and (S)-12, respectively.
Deprotection of 12 gave the corresponding (R)- and (S)-9 in
8.1% and 16% yield, respectively. Here, the yields were not
optimized. The structures of (R)- and (S)-7, 9, 11, and 12
In summary, we have developed a simple and practical
synthesis of (S)-N-substituted 4-hydroxy-pyrrolidin-2-ones
by hydroxylation of the corresponding pyrrolidin-2-ones with
Sphingomonas sp. HXN-200.
1
were identified by H and 13C NMR and MS spectra.
The ee of the biohydroxylation products 7 and 9 were
determined by HPLC with a chiral column:22 >99.9% ee
(S) for 7 and 90-92% ee (S) for 9, as shown in Table 3.
Acknowledgment. We are indebted to Mr. H.-J. Feiten
(ETH Zurich) for preparing cells of Sphingomonas sp. HXN-
200. We thank Dr. J. B. van Beilen (ETH Zurich) for helpful
discussion and Mr. Th. Plaggemeier and Prof. K. Engesser
(University of Stuttgart) for supplying us with the HXN-
200 strain.
Table 3. Preparation of 7 and 9 by Hydroxylation of 6 and 8,
Respectively, with Resting Cells of Sphingomonas sp. HXN-200
substrate
(mM)
cells
(g/L)
conversion
(%)
yield
(%)
ee (S)
Supporting Information Available: Experimental details
for biocatalytic preparation of 9; data of chemically prepared
product
(%)
6 (3.0)
6 (6.0)
6 (6.0)
8 (14)
8 (14)
8 (20)
4.0
8.0
10
4.0
8.0
10
7
7
7
9
9
9
66
59
75
48
66
49
55
42
68
39
46
42
>99.9
>99.9
>99.9
90
92
90
1
compounds 7, 9, 11, and 12; H and 13C NMR spectra of
biohydroxylation products 7 and 9. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL006735Q
(22) (a) The ee of 7 was determined by analytical HPLC: column,
Chiralpak AS.; eluent, n-hexane/2-propanol 4:1; flow, 1.0 mL/min; TR (S)
) 20.3 min; TR (R) ) 30.5 min. (b) The ee of 9 was determined by analytical
HPLC: column, Chiralcel OB-H; eluent, n-hexane/2-propanol 7:1; flow,
0.5 mL/min; TR (R) ) 17.9 min; TR (S) ) 22.6 min.
The ee of 9 was increased from 92% to 99.9% (S)20c in
82% yield by simple crystallization from n-hexane/ethyl
acetate (2:1).
3952
Org. Lett., Vol. 2, No. 24, 2000